1. Field of the Invention
Gram-negative bacterial disease and its most serious complications, e.g., bacteremia and endotoxemia, are the cause of significant morbidity and mortality in human patients.
An examination of many of the cases documented in the literature indicates that some of the manifestions of infection with gram-negative bacteria, especially lethal manifestations are associated with certain predisposing factors. There is an increased incidence of such infections in elderly patients and in patients who have serious underlying medical conditions such as burns, surgical trauma, slow-healing wounds, narcotic addiction, or malignancies. These infections may be of nosocomial (i.e., hospital-acquired) origin in patients who have sustained prolonged hospitalization, and particularly in patients who have been subjected to surgical intervention, intravascular instrumentation, or manipulative procedures such as urethral catheterizations, cystoscopies, tracheostomies, lumbar puncture, and intravenous infusion of medications and fluids or who have been placed on long-term therapy with immunosuppressive agents, corticosteroids, antimetabolites, and/or antibiotics. Radiation therapy also predisposes a mammalian host to infection by gram-negative bacteria.
Included amoung the most frequently encountered organisms in gram-negative disease are Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Pseudomonas aeruginosa, Serratia marcescens, and various species of Proteus, Bacteroides, Providencia, and Citrobacter (Sonnenwirth, "The Enteric Bacilli and Similar Gram-Negative Bacteria," pp. 753-790, Microbiology, 2nd edition, Davis, B. D., Dulbecco, R., Eisen, H. N., Ginsberg, H. S., Wood, W. B., and McCarty, M., eds., Harper and Row (1973); McCabe, W. R., "Gram-Negative Bacteremia," Adv. Intern. Med. (1974) 19:135-158; and Kreger et al., "Gram-Negative Bacteremia III. Reassessment of Etiology, Epidemiology and Ecology in 612 Patients," Am. J. Med. (1980) 68:332-343). Also, other species of Pseudomonas and Klebsiella as well as members of the genera Aeromonas, Salmonella, Flavobacterium, Erwinia, Edwardsiella, Pectobacterium, Acinetobacter, Alcaligenes, and Shigella contribute to a significant proportion of gram-negative disease in humans (Sonnenwirth, supra; McCabe, supra; Kreger et al supra).
During the past few decades, antibiotics have been the therapy of choice in the control of gram-negative bacterial disease. The continued prevalence and high morbidity and mortality associated with gram-negative bacterial disease, however, suggest limitations of antibiotic therapy to prevent and treat disease by these organisms. See, for example, Andriole, V. T., "Pseudomonas Bacteremia: Can Antibiotic Therapy Improve Survival?", J. Lab. Clin. Med. (1978) 94:196-199. This has prompted the search for alternative prevention and treatment methods.
2. Description of the Prior Art
Active immunization of man or experimental animals with whole bacterial cell vaccines or purified bacterial endotoxins leads to the development of specific antibodies directed mainly against the chemically diverse repeating oligosaccharide determinants present on lipopolysaccharide (LPS) molecules (Luderitz et al., "Immunochemistry of O and R Antigens of Salmonella and Related Enterobacteriaceae," Bacteriol. Rev. (1966) 30:192-255; and Luderitz et al., "Isolation and Chemical and Immunological Characterization of Bacterial Lipopolysaccharides," Microbial Toxins, Vol. 4, pp. 145-233, Weinbaum, G., Kadis, S., and Ajl, S. J., eds., Academic Press (1977), see FIG. 1). In this regard, it is important to note that LPS molecules are one of the major constituents of the outer cell membrane of most, if not all, gram-negative bacteria (Nikaido, H., "Biosynthesis and Assembly of Lipopolysaccharide," Bacterial Membranes and Walls, Leive, L., ed., Marcel Decker, (1973); see FIG. 2). In FIG. 1 is illustrated a model of the gram-negative cell wall showing the position of lipopolysaccharide molecules with respect to the outer membrane of the bacterium. This model is devoid of structures such as capsules, envelopes and slime layers.
Lipopolysaccharides are also integral constituents of bacterial endotoxins, with the lipid A moiety of LPS molecules being responsible for the profound pathophysiologic effects (e.g., fever, hypotension, disseminated intravascular coagulation, shock, and potentially death) associated with endotoxin release during gram-negative bacterial septicemia or induced experimentally with injections of endotoxin (Westphal et al., "Chemistry and Immunochemistry of Bacterial Lipopolysaccharides as Cell Wall Antigens and Endotoxins," Prog. Allergy (1983) 33:9-39). FIG. 2 illustrates the three regions of gram-negative lipopolysaccharides. The O-specific chain region is a long-chain polysaccharide built up from repeating oligosaccharides units. In different bacterial species these oligosaccharide units may contain from 1 to as many as 6 or 7 monosaccharide units. Serotypic determinants of LPS (see text) are displayed on this region of the molecule. The core region contains several monosaccharides in arrangements that are relatively invariant from one strain of gram-negative bacteria to another. The lipid A region is virtually the same in all gram-negative bacteria, is generally attached to the core region through the keto-deoxyoctonic acid moiety, and serves as the attachment point of the lipopolysaccharide molecule to the outer membrane.
The induction and immunotherapeutic use of type-specific anti-LPS antibodies have been most extensively studied in the treatment of disease due to Pseudomonas aeruginosa (P. aeruginosa) because of its high degree of antibiotic resistance. Such antibodies, whether actively engendered or passively transferred, have been shown to be protective in a variety of animal infection models. For a review, see Pollack, M., "Antibody-Mediated Immunity in Pseudomonas Disease and Its Clinical Application," Immunoglobulins: Characteristics and Uses of Intravenous Preparations, Alving, B. M. and Finlayson, J. S., eds., pp. 73-79, U.S. Department of Health and Human Services, (1979).
Perhaps more importantly, high acute serum titers of antibodies to the type-specific portions of LPS molecules of infecting strains have been observed to be associated with survival in patients with P. aeruginosa bacteremia (Pollack, M. and Young, L. S., "Protective Activity of Antibodies to Exotoxin A and Lipopolysaccharide at the onset of Pseudomonas aeruginosa Septicemia in Man," J. Clin. Invest. (1979) 63:276-286). This observation has, in fact, been found to extend to the majority of bacteremias caused by various gram-negative organisms (Zinner, S. H. and McCabe, W. R., "Effects of IgM and IgG Antibody in Patients with Bacteremia Due to Gram-Negative Bacilli," J. Infect. Dis. (1976) 133:37-45; and Clumeck et al., "Humoral Immunity and Circulating Immune Complexes in Gram-Negative Bacteremia and Septic Shock," Bacterial Endotoxins and Host Response, pp. 79-94, Agarwal, M. K., ed., Elsevier, (1980)).
Although the precise means by which anti-LPS antibodies exert such protection have not been entirely delineated (particularly with respect to the various genera of gram-negative bacteria), it is generally thought that they do so by faciliating clearance of LPS molecules from the bloomstream by the reticuloendothelial system or by rendering LPS containing bacteria sensitive to complement-mediated lysis and/or phagocytosis (Morrison, D. C. and Ryan, J. L., "Bacterial Endotoxins and Host Immune Responses," Adv. Immunol. (1979) 28:293-450). Conceivably, any one or a combination of the above protection schemes may be at work in serious gram-negative bacterial disease.
In 1975 Kohler and Milstein reported their discovery that certain mouse cell lines could be fused with mouse spleen cells to create hybridomas which would secrete pure "monoclonal" antibodies (Kohler, G., and Milstein, C., "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature (1975) 256:495-497).
In a 1982 abstract (#253) published on p. 110 of the Abstracts of the 1982 Interscience Conference on Antimicrobial Agents and Chemotherapy, J. Sadoff et al., reported the production, via hybridoma techniques, of mouse (murine) monoclonal antibodies of the IgM class directed against the oligosaccharide determinants of the LPS molecules of a particular strain (serotype) of P. aeruginosa and that these murine monoclonal antibodies provided protection to mice against a lethal challenge of P. aeruginosa bacteria of the same strain (i.e., the homologous strain).